Vol. 16(32), pp. 1699-1704, 9 August, 2017 DOI: 10.5897/AJB2017.16086 Article Number: 28412E265692 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB

Full Length Research Paper

Antibacterial activity of Aspergillus isolated from different Algerian ecosystems

Bramki Amina1*, Ghorri Sana1, Jaouani Atef2, Dehimat Laid1 and Kacem Chaouche Noreddine1

1Laboratory of Mycology, Biotechnology and Microbial Activity, University of Mentouri Brothers- Constantine, P.O. Box, 325 Ain El Bey Way, Constantine, Algeria. 2Laboratory of Microorganisms and Active Biomolecules, University of Tunis El Manar, Campus Farhat Hached, B.P. no. 94 - Rommana, Tunis 1068, Tunisia.

Received 26 May, 2017; Accepted 4 August, 2017

Thirty two strains of Aspergillus genus were isolated from soil samples obtained from particular ecosystems: Laghouat endowed with a desert climate and Teleghma with a warm and temperate climate. Based on the morphological aspect, this collection was subdivided into ten phenotypic groups. This identification was confirmed by molecular analyzes using a molecular marker of the genu ribosomal 18s. This marker will allow us to associate our sequences with those of known organisms. In order to discover new antibiotic molecules, the antibacterial activity was performed against two Gram positive bacteria: Staphylococcus aureus and Bacillus subtilis and also two Gram-negative bacteria: Escherichia coli and Pseudomonas aeroginosa, using two different techniques: Agar cylinders and disks technique. The results show that the fungal species have an activity against at least one test bacterium. The Gram positive bacteria were the most affected, where the averages of the inhibition zones reach 34.33 mm. However, Gram-negative bacteria showed less important results from 0 to 12.00 mm. It is recorded that the antibacterial activity was studied for the first time in the following two species: Aspergillus niveus and Aspergillus wentii. Furthermore, an indepth study is underway on bioguided fractionation, which would identify individual components and lead to the isolation of the active ingredient.

Key words: Aspergillus, particular ecosystems, antibacterial activity.

INTRODUCTION

The massive use of antibiotics has not led to the making multi-resistant strains to be responsible for elimination of infections, but has made microbes resist serious infections. In recent years, bacterial resistance to these antibiotics by exerting a selection pressure which antibiotics has become a worrying global phenomenon favors the emergence of resistance genes in bacteria, (Courvalin and Philippon, 1990; Bevilacqua, 2011). Due

*Corresponding author. E-mail: [email protected]. Tel: +213 6 69 43 31 06.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1700 Afr. J. Biotechnol.

to the increase in infections involving multi-resistant followed by successive subcultures in the same isolation medium bacteria, the need for new effective antibacterial (Boudoudou et al., 2009), and they were preserved in the form of treatments is more and more imperious (Savin, 2014). spores in a glycerol/saline solution of 30% at -20°C (Botton et al., 1990). For this, the biodiversity is exploited. The isolation of strains from rarely exploited ecosystems allows the discovery of strains that may have a high potential or Morphological identification unexploited production. In addition, the use of antibiotic- resistant bacteria as germs tests can lead to the The strain identification was performed by macroscopic observation (growth rate, colonies color and color variation over time, colonies discovery of effective molecules which may be new upside color, surface texture, etc) and microscopic characters (Boughachiche et al., 2012). In this field, fungi have been (mycelium, conidiophores, conidiogenesis, conidia, etc) (Botton et widely studied. Of a total of 10700 antibiotics described al., 1990; Chabasse et al., 2002, 2008). for the entire living world, about 1600 are from fungi (Botton et al., 1990). Aspergillus species are ubiquitous molds, widely Molecular identification distributed in nature (Bennett et al., 2010). From an DNA extraction and quantification economic view, the species of this genus have a great ecological and medical importance. The production of First, a quantity of mycelium (≈ 100 mg) was mashed in the extracellular and intracellular secondary metabolites by presence of 500 µl of lysis buffer (400 Mm Tris/HCl, 60 Mm EDTA, several species of this genus was detected in Aspergillus 150 mM NaCl, 1% SDS, 2 ml H2O ultrapure). The mixture was left aculeatus, Aspergillus ochraceus and Aspergillus terreus. to rest for 10 min and then 150 µl of potassium acetate was added (pH 4.8, solution prepared from 60 ml of a potassium acetate The latter are considered to be the highest producers of solution 5 M, 11.5 ml glacial acetic acid and 28.5 ml milliQ water). extracellular secondary metabolites (Youcef-Ali, 2014). After that, the tube was homogenized using a vortex, and Indeed, Aspergillus are known for their ability to produce centrifuged for 10 min at 10000 rpm, and the supernatant was antibacterial substances such as A. flavus used in the transferred to another tube of 1.5 ml volume on which an equal manufacturing of aspergillic acid, A. ochraceus used in volume of isopropanol was added. Then, the tube was agitated by inversion (10 times) and the manufacturing of penicillic acid and A. Fumigatus incubated at 20°C overnight. After incubation and centrifugation used in the manufacturing of fumagillin (Botton et al., (10000 rpm for 10 min), the supernatant was thrown and the DNA 1990; Taniwaki et al., 2003; Abdelaziz, 2006). pellet was rinsed twice with 300 µl of 70% ethanol. After This study aimed to show the antibacterial activity of centrifugation (10000 rpm for 10 min), the supernatant was Aspergillus strains isolated from different ecosystems and removed and dried in the hood for 2 h. The DNA was dissolved in producers of active metabolites used against Gram 50 µl of TE (Tris-EDTA). The DNA assay was realized by spectrophotometer (Thermo positive and negative resistant bacteria through different Scientific NanoDrop 2000) at two different wavelengths: the methods. absorption wavelength of nucleic acids (260 nm), and the absorption wavelength of proteins (280 nm). Then the DNA samples were stored at -20°C for further use (El Khoury, 2007). MATERIALS AND METHODS

Sampling PCR amplification of internal transcribed spacer (ITS) regions of rDNA This work deals with the isolation of Aspergillus from soil. For this purpose, the sampling was done in two different Algerian regions The Internal Transcribed Spacer (ITS) region of the ribosomal DNA namely: Laghouat (located 400 km in south Algeria at 750 m of was amplified with universal primers ITS1 (5’- altitude) and Teleghma (located in north - eastern Algeria). TCGGTAGGTGAACCTGCGG-3’) and ITS4 (5’- To do this, 100 g of soil was sampled up to 20 cm deep after TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). For isolates, discarding the first three centimeters of soil, and then deposited in PCR was performed in a total reaction volume of 25 μl containing 5 glass bottles under strict aseptic conditions. Mycological analysis μl of 5X buffer (Promega), 1 μl of dNTP (20 mM), 1.5 μl of MgCl2 was done on arrival at the laboratory (Pochon, 1964; Almi et al., (25 mM), 0.25 U (5 U/μl) Taq DNA polymerase, 2 μl of each primer 2015). (20 pmoles/μl), 11.25 µl of ultrapure H2O, and 2 μl of genomic DNA (50 ng/μl). The amplification program included an initial denaturation at 95°C Isolation for 3 min, followed by 35 cycles of denaturation at 98°C (15 s), annealing at 59°C (60 s), extension at 72°C (120 s) and a final The isolation was performed by the suspension - dilution method extension period of 10 min at 72°C in a Biometra thermal cycler (Davet, 1996; Davet and Rouxel, 1997). Decimal dilutions were (Germany). prepared from 10-1 to 10-6. Then, 0.1 ml of each suspension is rolling out on the surface of a potato dextrose agar (PDA: 200 g of potato, 20 g sugar, 20 g agar and 1000 ml distilled water) medium. Sequencing Three dishes were prepared for each dilution, and the plates were incubated at 28°C for 6 days. After an electrophoresis on 1.5% agarose gel of the amplification The proliferation of bacteria was prevented by adding an products, an enzymatic sequencing of DNA fragments was antibiotic, streptomycin (5 mg/l) in the culture medium (Botton et al., performed according to the method of incorporation of 1990). After the appearance of fungal colonies, these were purified dideoxynucleotide terminators system (Sanger et al., 1977). After Bramki et al. 1701

purification and quantitation, a precise aliquot of DNA was used as technique, DMSO was used as negative control. It should be noted a template of an enzymatic amplification reaction in the presence of that four repetitions were performed for each technique. And the dideoxynucleotides (ddNTP's) labeled with Big Dye. diameters of the inhibition zones were measured in millimeter.

Sequence comparison with the data bank RESULTS AND DISCUSSION

Sequence comparison with those of the databases was performed using the BLAST algorithm (Altschul et al., 1997). The alignments Screening has always been the key to achieving new of the nucleotide sequences were realized with the software antibacterial molecules. Although its performance has Chromas (Larkin et al., 2007). been reduced in recent years, it is been practiced till date in many laboratories. They have endeavored to diversify

Antibacterial activity of isolated Aspergillus species the sources of microorganisms by developing selection methods that favor new species (Le Berre and All the isolated species were tested for their antibacterial activity. Ramousse, 2003). For that purpose, two techniques were used: the agar cylinders and For this purpose, our work focuses on the research of disc technique. antibacterial activity of certain strains of Aspergillus genus isolated from the soil sampled from particular

Preparation of microorganisms test ecosystems: Laghouat has a desert climate and Teleghma has a warm and temperate climate. Indeed, The antibacterial activity of isolated Aspergillus was sought against the soils of these zones, which constitute virtually ATCC test bacteria (American Type Culture Collection), which are particular media, seem to be promoter environments for Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633) the isolation of fungi producing new antibacterial Escherichia coli ATCC 25922, Pseudomonas aeroginosa ATCC substances. After isolation and purification, 32 strains of 9027.The reactivation of the bacterial strains was carried out by seeding on selective media; Chapman, TSA (Trypticase Soy Agar), the genus Aspergillus were obtained from all samples. Hecktoen, and cetrimide agar, respectively. Based on the morphological characteristics, the strains Bacterial suspensions were prepared from the 18 h cultures. The collected were subdivided into 10 phenotypic groups. cell density of each suspension was adjusted by dilution in sterile Identification standards are mainly morphological. physiological water, and in comparison with the 0.5 McFarland However, the application of molecular characterization solution (An optical density equal to 0.2 at 650 nm) in order to obtain a final concentration of 106 CFU/ml (Cavalla and Eberlin, tools has shown that this strictly phenotypic identification 1994). could lead to misidentification and that certain species groupings have no foundation (Thierry, 2011). For this, this morphological identification was confirmed by The agar cylinders technique molecular analyzes. The encoding DNA of 18S ribosomal RNAs of the Aspergillus strains were seeded on PDA medium. After 14 days of incubation at 28°C, agar cylinders of 6 mm diameter were removed isolates, extracted and amplified, is separated by agarose and deposited on the Mueller-Hinton medium surface previously gel electrophoresis. The DNA bands obtained correspond seeded with the test bacteria. The dishes were then placed at 4°C to that of 600 base pairs of the molecular weight marker. for 4 h to allow diffusion of the active substances, and then The PCR products obtained were sequenced and incubated at 37°C for 18 to 24 h (Tortorano et al., 1979; Gungi et subsequently compared with the sequences of the other al., 1983). microorganisms recorded in the Genbank database

(Table 1). Preparation of extracts GenBank, through the Blastn program, realizes an alignment and proposes a sequence that presents the Aspergillus strains were reseeded in the potato dextrose broth best bits of score and percent identity with that of the (PDB) medium. After 14 days of incubation at 28°C, the formed biomass was removed by filtration, then the filtrate obtained was current study. The 10 strains aligned were identified as added to an equal volume of chloroform. After decantation, the follows: Aspergillus fumigatus, Aspergillus niveus, chloroform phase was concentrated by vacuum evaporation using a Aspergillus wentii, Aspergillus fumigatiaffinis, Aspergillus Rotavapor (Gengan et al., 1999; Ghorri, 2015). Each fungal extract quadrilineatus, Aspergillus nidulans, Aspergillus terreus, was dissolved in dimethyl sulfoxide (DMSO) in order to obtain a Aspergillus flavus, Aspergillus sclerotiorum and concentration of 100 mg/ml. Aspergillus niger.

After isolation and purification, the fungal species were Discs technique screened on their antibacterial potency using two different techniques. The inhibition zones were measured Discs of 6 mm diameter of Whatman paper soaked with 10 μl of the after 24 h of incubation and the averages of these zones extract to be tested were dried and deposited on the surface of the are shown in Tables 2 and 3. For the agar cylinders dishes containing Mueller-Hinton medium previously inoculated with the test bacteria. Then, the dishes were incubated at 4°C for 2 h; technique, the results show that 9 out of 10 fungal thus the metabolites can diffuse, and then incubated at 37°C for 18 species have an antibacterial activity against S. aureus to 24 h (Yamaç and Bilgili, 2006; Hazalin et al., 2009). For this bacterium only and the averages diameter of the inhibition 1702 Afr. J. Biotechnol.

Table 1. Confrontation and biomolecular corresponding with GenBank.

Isolate Strains proposed by GenBank Score E-value Identity (%) L2 Aspergillus quadrilineatus 812 0.0 99 L3 Aspergillus fumigatus 977 0.0 100 L61 Aspergillus fumigatiaffinis 835 0.0 99 T1 Aspergillus flavus 832 0.0 100 T5 Aspergillus niger 834 0.0 99 T7 Aspergillus nidulans 788 0.0 99 T27 Aspergillus terreus 877 0.0 100 T32 Aspergillus niveus 872 0.0 100 T33 Aspergillus wentii 754 0.0 99 T62 Aspergillus sclerotiorum 821 0.0 99

L, Laghouat ; T, Teleghma.

Table 2. Demonstration of the antibacterial activity of fungal species by agar cylinders technique.

Zone of inhibition (mm) Strain S. aureus B. subtilis E. coli P. aeroginosa L2 33.67 ± 6.03 - - - L3 34.33 ± 6.03 - - - L61 30.67 ± 1.15 - - - T1 13.00 ± 2.00 - - - T5 - - - - T7 16.67 ± 1.15 - - - T32 33.00 ± 2.65 - - - T33 31.33 ± 3.21 - - - T46 32.00 ± 2.00 - - - T62 30.67 ± 1.15 - - -

*Diameter of the inhibition zone ≤ 6 mm.

Table 3. Demonstration of the antibacterial activity of fungal species by disks technique.

Zone of inhibition (mm) Strain S. aureus B. subtilis E. coli P. aeroginosa L2 16.67 ± 1.15 10.33 ± 1.53 11.00 ± 1.00 - L3 31.67 ± 1.53 30.00 ± 4.36 7.67 ± 0.58 - L61 16.33 ± 1.15 11.00 ± 1.00 8.67 ± 0.58 - T1 7.00 ± 0.00 9.00 ± 1.00 8.67 ± 0.58 - T5 7.33 ± 0.58 9.67 ± 1.53 9.00 ± 0.00 - T7 12.00 ± 1.00 9.33 ± 0.58 10.33 ± 0.58 - T32 18.33 ± 1.53 25.00 ± 3.00 11.33 ± 0.58 8.00 ± 1.00 T33 11.33 ± 0.58 12.00 ± 1.00 9.67 ± 0.58 7.00±1.00 T46 11.67 ± 1.15 12.67 ± 1.53 10.33 ± 2.08 - T62 7.00 ± 0.00 9.00 ± 0.00 - -

*Diameter of the inhibition zone ≤ 6 mm.

zones ranged from 13 to 34.33 mm. A. terreus, A. wentii, A. fumigatiaffinis and A. sclerotiorum The species A. fumigatus, A. quadrilineatus, A. niveus, showed an important antibacterial activity with Bramki et al. 1703

respectively 34.33, 33.67, 33.32, 31.33, 30.67, 30.67 mm (Kumara et al., 2010). of inhibition averages (Table 2). After extraction with chloroform, the metabolic extracts of the 10 fungal species were examined for their antibacterial activity by Conclusion disks technique. The various extracts showed a more or less The ten strains of Aspergillus isolated in this study considerable antibacterial activity where the averages of showed a considerable antibacterial activity. It is the inhibition zones ranged from 7 to 31.67 mm, for the S. interesting to test them against a range of human aureus bacterium, from 9 to 30 mm, for B. subtilis and pathogens. Furthermore, an in-depth study is underway from 0 to 11.33 mm for E. coli unlike P. aeroginosa on on bio-guided fractionation, which would identify which the extracts of all species had no effect except the individual components and lead to the isolation of active metabolic extracts of the A. niveus and A. wentii species ingredients. that gave inhibition zones of 8 and 7 mm diameters, respectively. This technique revealed that the majority of species present antibacterial activity on at least one of CONFLICT OF INTERESTS the test bacteria and the species A. fumigatus, A. niveus, A. quadrilineatus and A. fumigatiaffinis showed The authors have not declared any conflict of interests. considerable antibacterial activity (Table 3). In fact, the species of Aspergillus genus are known by REFERENCES their production of substances having an antibacterial effect (Maria et al., 2005; Madki et al., 2010). These Abdelaziz W (2006). Isolement des mycètes producteurs des biologically active secondary metabolites are synthesized substances antibactériennes à partir des sols sahsriens. UFMC. at the end of the growth (Attalah and Kacem-chaouche, http://www.secheresse.info/spip.php?article27315. Almi H, Dehimat L, Kacem chaouche N (2015). The pattern of pathogen 1992). Irobi et al. (2000) worked on the A. quadrilineatus diversity and abundance in Lentil (Lens culinaris) fields in fungal species and found that these species have Constantine region, Algeria. Afr. J. Agric. 10(13):1536-1542. remarkable antimicrobial activity against S. aureus and B. Al-Shaibani A B A, Al-Shakarchi FI, Ameen R S (2013). Extraction and subtilis; Niege et al. (2002) showed that A. fumigatus Characterization of Antibacterial Compound from Aspergillus niger. J. Al-Nahrain Univ. 16(4):167-174. have a remarkable antimicrobial activity against S. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, aureus, Candida albicans and Micrococcus luteus. Lipman DJ (1997). Gapped BLAST and PSI-BLAST: a new Furthermore, Barakat and Gohar (2012) showed that A. generation of protein database searchprograms. Nucleic Acids Res. terreus has a considerable antibacterial activity as 25:3389-3402. Attalah M, Kacem Chaouche N (1992). Production of ochratoxin A in a compared to certain classic antibiotics; Al-Shaibani et al. semisynthetic. In: The second Regional Mycoligical Conference RMC (2013) worked on the antibacterial activity of A. niger 2, Cairo. Egypt. fungal species. The results revealed that this species has Barakat KM, Gohar YM (2012). Antimicrobial Agents Produced by an inhibitory effect against P. aeruginosa, S. aureus, S. Marine Aspergillus terreus var. africanus Against Some Virulent Fish Pathogens. Indian J Microbiol. 52(3):366-372. epidermidis, and Bacillus sp. The same results were Bennett TW, Machida M, Gomi K (2010). On overview of the genus observed by Ola et al. (2014) who found that neosartorin Aspergillus. In. Aspergillus. Molecular Biology and Genomics. pp. 1- isolated from A. fumigatiaffinis had an important 19. antibacterial activity against a large spectrum of Gram- Bevilacqua S (2011). Evaluation de l’impact d’une équipe opérationnelle en infectiologie sur la consommation et le cout des antibiotiques au positive bacterial species, including Staphylococci, CHU de Nancy, (Essai d’intervention contrôlé). SVS. Université Streptococci, Enterococci and B. subtilis. In addition, Nancy, France. pp. 18-25. Phainuphong et al. (2017) found that penicillic acid Botton B, Bretton A, Fever M, Gautier S, Guy Ph, Larpent JP, Reymond isolated from A. sclerotiorum has antibacterial activity P, Sanglier J-J, Vayssier Y, Veau P (1990). Moisissures utiles et nuisibles, importance industrielle, (edn) Masson, Paris. against S. aureus and E. coli with MIC values of 128 Boudoudou H, Hassikou R, Ouazzani touhami A, Badoc A, Douira A mg/mL. The antibacterial activity against Gram positive (2009). Paramètres physicochimiques et flore fongique des sols de bacteria tests appears to be more important than the one rizières Marocaines. Bull. Soc. Pharm. Bordeaux 148:17-44. against Gram negative staining bacteria. This is related to Boughachiche F, Reghioua S, Zerizer S, Boulahrouf A (2012). Antibacterial activity of rare Streptomyces species against clinical the results obtained by Prabavathy and Nachiyar (2012). resistant bacteria. Ann. Biol. Clin. 70(2):169-174. These results can be explained by the fact that these Cavalla M, Eberlinberlin T (1994). Isolement des streptomycetes du sol. two groups of microorganisms differ morphologically, l’opéron 19:13-17. because Gram negative bacteria have an outer Chabasse D, Bouchara JP, Gentile L, Brun S, Cimon B, Penn P (2002). Les moisissures d’intérêt médical. (edn) Bioforma. Paris. 160 p. membrane which is a polysaccharide membrane carrying Chabasse D, Contet-audonneau N, Bouchara J P, Basile AM (2008). the lipopolysaccharide structural components. This Moisissures dermatophytes levures du prélèvement au diagnostic. makes the cell wall impermeable to lipophilic compounds, Ed. bioMérieux, France. unlike Gram-positive bacteria, which will be more Courvalin P, Philippon A (1990). Mécanismes biochimiques de la résistance bactérienne aux agents antibactériens. Bactériologie sensitive because they only have an outer peptidoglycan médicale. Medecine- Sciences. Flammation. France. Ch 14:332-355. layer, which is not an effective permeability barrier Davet P (1996). Vie microbienne du sol et production végétales, (edn) 1704 Afr. J. Biotechnol.

INRA. Paris. Pochon J (1964). Manuel technique d'analyse microbiologique. Masson Davet P, Rouxel F (1997). Detection et isolation des champignons du et Cie. Paris (Ed.). pp. 5-20. sol, (edn) INRA. Paris. Prabavathy D, Valli Nachiyar C (2012). Study on the antimicrobial El khoury A (2007). Champignons Mycotoxinogènes et Ochratoxine A activity of Aspergillus sp isolated from Justicia adathoda. Indian J. (OTA) et Aflatoxine B1 (AFB1) dans les vignobles libanais: Sci. Technol. 5(9):974-6846. Occurrence et Origine. L’institut National Polytechnique De Toulouse. Sanger F, Nicklen S, Coulson AR (1977). DNA sequencing with chain- Gengan RM, Chuturgoon AA, Mulholland DA, Dutton MF (1999). terminating inhibitors. Proc. Natl. Acad. Sci. 74:5463-5467. Synthesis of sterigmatocystin derivatives and their biotransformation Savin BM (2014). La phagothérapie : historique et potentielle utilisation to aflatoxins by a blocked mutant of Aspergillus parasiticus. contre les infections à bactéries multirésistantes. Faculté de Mycopathologia 144:115-122. médecine de créteil. Ghorri S (2015). Isolement des microorganismes possédant une activité Taniwaki MH, Pitt JI, Teixeira AA, Iamanaka BT (2003). The source of anti- Fusarium. UFMC. ochratoxin A in Brazilian coffee and its formation in relation to Gungi S, Arima K, Beppy T (1983). Screening of antifungal antibiotics processing methods. Int. J. Food. Microbiol. 82:173-179. according to activities inducing morphological abnormalities. Agric. Thierry S (2011). Etude de la diversité génétique et du pouvoir Biol. Chem. 47:2061-2069. pathogène d’Aspergillus fumigatus et de Chlamydophila psittaci chez Hazalin NA, Ramasamy K, Lim SM, Wahab IA, Cole AL, Abdul Majeed les oiseaux. L’Institut des Sciences et Industries du Vivant et de AB (2009). Cytotoxic and antibacterial activities of endophytic fungi l’Environnement. isolated from plants at the National Park, Pahang, Malaysia. BMC Tortorano AM, Cabrini E, Viviani MA (1979). Sensibilité in vitro des Complement. Altern. Med. 9:46. levures a cinq antibiotiques. Comparaison de deux méthodes CMI en Irobi ON, Gbodi TA, Moo-Young M, Anderson WA (2000). Antibiotic gélose et méthode des disques. Bull. Soc. Fr. Myc. Med. 8:69-74. activity of Aspergillus quadrilineatus extracts isolated from a Nigerian White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct cereal. Pharm. Biol. 38(1):57-60. sequencing of fungal ribosomal RNA genes for phylogenetics. In. Kumara CG, Mongollaa P, Josepha J, Nageswara YVD, Kamal A PCR protocols: a guide to methods and applications, Academic (2010). Antimicrobial activity from the extracts of fungal isolates of Press, San Diego. pp. 315-322. soil and dung samples from Kaziranga National Park, Assam, India. Yamaç M, Bilgili F (2006). Antimicrobial Activities of Fruit Bodies and/or J. Med. Mycol. 20:283-289. Mycelial Cultures of Some Mushroom Isolates. Pharm. Biol. 44:660- Larkin MA, Blackshields G, Brown NP, Cenna R, McGettigan PA, 667. McWilliam H (2007). Clustal W and Clustal X version 2.0. Youcef-Ali M (2014). Etude de l’activité anti-Candida albicans des Bioinformatics 23(21):2947-2948. microorganismes isolés à partir du sol des zones arides. UFMC. Le Berre M, Ramousse R (2003). Les enjeux de la conservation de la biodiversité en milieu saharien. Socioloécologie et Conservation. Université Claude Bernard Lyon1: 18p. Madki M A, Manzoor A S, Powar P V, Patil K S (2010). Isolation and Biological Activity of Endophytic Fungi from Withania Somnifera. Int. J. Pharm. Sci. 2:848-858. Maria G L, Sridhar K R, Raviraja N S (2005). Antimicrobial and enzyme activity of mangrove endophytic fungi of southwest coast of India. J. Agric. Technol. 1:67-80. Niege AJCF, Suraia S, Izabel YI, Jairo KB (2002).The antimicrobial activity of Aspergillus fumigatus is enhanced by a pool of bacteria. Microbiol. Res. 157:207-211. Ola ARB, Debbab A, Aly AH, Mandi A, Zerfass I, Hamacher A, Kassack MU, Brötz-Oesterhelt H, Kurtan T, Proksch P (2014). Absolute configuration and antibiotic activity of neosartorin from the endophytic fungus Aspergillus fumigatiaffinis. Tetrahedron Lett. 55(5):1020-1023. Phainuphong P, Rukachaisirikul V, Tadpetch K, Sukpondma Y, Saithong S, Phongpaichit S, Preedanon S, Sakayaroj J (2017). ɣ- Butenolide and furanone derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Phytochemistry 137:165- 173.